Objective

Deterioration in sperm quality with age is a major cause of male infertility. This has particular socio-economic relevance for Europe where fatherhood is increasingly delayed and infertility has reached near epidemic levels, costing the EU €4 billion per annum. The mechanisms causing, and the full consequences of declining sperm quality with age remain very poorly understood. For example, it is unclear how much of the reduction in sperm quality is due to advanced male age or the time sperm are stored within males reproductive organs, and we do not understand the mechanisms responsible for deteriorating sperm quality with increasing male age. The general aim of this project is to disentangle the effects of male aging and sperm aging on sperm quality, fertility and reproductive fitness using Drosophila melanogaster as a model. We will also test putative mechanisms thought to cause age-related fertility declines and test two possible general pathways through which sperm age. This proposal is a much needed integration of evolutionary ecology and sperm physiology, benefiting from the ideal match of skills and expertise of the Applicant (Tourmente - physiology), and the PI (Hosken - evolution). Since the mechanisms and phenomena studied are conserved across animals, the research proposed has high potential to produce results relevant to fundamental and applied research in human aging and reproductive health and animal breeding. The MSC fellowship will be pivotal for Applicants development toward independent research and offers the unique opportunity to work in one of the most prominent evolutionary ecology groups worldwide. The Biosciences Department at the University of Exeter is world-leading and provides excellent opportunities for the personal career development of the Applicant, who in turn brings novel skills to Exeter that will greatly benefit this group.

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It has been said the West is undergoing a male fertility crisis with recent research from the Hebrew University in Israel showing that sperm counts among men have more than halved in the past 40 years. The SPERMAGE projects set out to look for clues as to why.

Declining sperm quality with age is a major cause of male infertility and a problem in many parts of the world including Europe where it has particular relevance as fatherhood is increasingly delayed.
Yet there remains poor understanding of both the underlying mechanisms which cause sperm to deteriorate, along with inadequate appreciation of the full consequences of these processes. For example, it is unclear how much of the sperms’ reduced quality is due to advanced male age or the time sperm are stored within male reproductive organs. What is known is that livestock have not undergone a similar decline.
The EU-funded Marie Skłodowska-Curie Fellowship project, SPERMAGE, set out to untangle the influence of male ageing and sperm ageing on fertility, reproductive fitness and sperm quality using fly (Drosophila) models.
The project found a strong genetic basis to sperm viability and some indication that male ejaculates with a high proportion of viable sperm may sometimes negatively impact female fertility.

Investigating the puzzle of sperm viability variation

The SPERMAGE team collectively brought more than 50 years of reproduction expertise to investigate some of the elements contributing to the puzzle of variation in sperm viability – put simply, why do males not consistently produce high quality ejaculates?
The team looked at Drosophila, the genus of fly widely used as models to investigate a range of biological phenomena. These flies are perfect for studies like this as they are easy to rear in large numbers, have short generations and share common genetic pathways with most animals.
The team established standardised genotypes by selectively breeding generations in the lab and then investigating the effects of genotype on sperm viability. They assessed the proportion of live and dead sperm in the testes of flies using a staining technique that marks live and dead sperm differently (live sperm stained green, dead stained red) and then tested whether differences in the proportion of live and dead sperm depended on genotype.
“Our work found some immediate causes of sperm quality decline and also possible ultimate reasons. For example, while viable sperm benefits competing males, high viability ejaculates may actually cause egg wastage in females by over-stimulating the female reproductive tract for example. So there may be evolutionary trade-offs underway in the fertility process,” says project coordinator, David Hosken.

Further down the line

Currently SPERMAGE’s results contribute chiefly to the EU’s knowledge and skills base. “As we were exploring highly novel areas of research we had to overcome some pretty difficult experimental challenges through intelligent experimental design. One major issue was obtaining enough sperm that we could then isolate and subject to experimental scrutiny,” says Hosken.
In the future, as the processes studied are comparable across animals, the results will become increasingly relevant to fundamental and applied research in human ageing, reproductive health and animal breeding.
The next step for the team will be to further investigate other possible causes of sperm inviability, along with how some of these underlying causes may interact, including testing putative mechanisms thought to cause age-related fertility declines.

Periodic Reporting for period 1 - SPERMAGE (No country for old sperm: male aging, sperm aging, and external stressors as determinants of sperm quality and fertility)

Human sperm quality declines have been documented in many countries and understanding the causes of these may help us develop better, cheaper male fertility treatments. This is a matter of enormous socio-economic concern for Europe, where nearly half of total infertility disorders are due to male factor alone, currently costing the EU ca. €4 billion per annum (www.bbc.co.uk/news/health-31754366). The causes, mechanisms, and consequences of declining sperm quality with age remain very poorly understood. First, it is unclear how much of the reduction in sperm quality is due to (a) declining sperm quality due to advanced male age, or (b) decreases in sperm performance as unused sperm age within males storage organs. Second, the consequences of sperm aging for male and female reproductive success are unclear. Finally, we have a limited understanding of the mechanisms responsible for deteriorating sperm quality with increasing male age. The general aim of this project is to disentangle the effects of male aging and sperm aging on sperm quality and fertility using fruit flies (Drosophila) as a model. Since the mechanisms and phenomena studied are conserved across animals, the research proposed has high potential to produce results relevant to fundamental and applied research in human aging and reproductive health, and animal breeding.

The overall objectives of this project are:

1. Determine whether sperm viability is genetically variable and heritable.2. Test how variations in ejaculate quality of young males affect female fitness.3. Identify the effects of sperm and male age on sperm competitiveness, number, and viability.4. Evaluate sperm and male aging effects on male and female reproductive success, and offspring health.5. Assess whether infection with a common parasite, Wolbachia, accelerates male aging and sperm aging.

We tackled objectives 1 and 2, a panel of Drosophila genotypes (isolines) that capture the genotypic variation of a population while facilitating the repeated measurement of the same genotype in a high number of individuals. We tested for the presence of genetic variation in sperm viability, i.e. the proportion of living sperm in the ejaculate, and its repeatability within the same genetic line (which sets the upper limit on the heritability of sperm viability). Furthermore, we mated tester females with genotypes from the extremes of the sperm viability range to assess how this trait affected female fitness. We discovered that while sperm viability is highly heritable and shows a high degree of variability between genotypes. In addition, we showed for the first time that high sperm viability increases female fitness by increasing the offspring numbers after single insemination (Tourmente et al., in revision). As noted above, there was also evidence of sexual conflict over sperm viability as genotypes producing more viable sperm caused females to waste more eggs despite generating higher female fitness overall.

To address objectives 3, 4 and 5 we performed a set of assays using males of 13 Drosophila isolines that were subjected to either, pre-meiotic sperm senescence (by aging the males), or post-meiotic sperm senescence (by manipulating the time since sperm depletion). In addition, some of the isolines were infected with Wolbachia which enabled testing of infection impacts on sperm viability. We discovered that genotype has a profound effect upon sperm both pre and post-meiotic sperm senescence. In other words, the genotype determines the direction and magnitude of the change in sperm viability with increased male aged and sperm storage time. Moreover, increased male age negatively impacts copulatory performance and male fertility, but results in higher quality daughters. Interestingly, however, neither sperm age nor parasite infection has an effect on these male reproductive traits. These findings are now in preparation for publication.

The knowledge gathered in the SPERMAGE project (the Fellow and his host group), reveal the main role of genotypic variance upon pre and post-meiotic sperm senescence. Since the mechanisms and phenomena studied are conserved across animals, this knowledge will inform further fundamental and applied research in human aging and reproductive health and animal breeding.

The prestigious Marie Skłodowska-Curie Actions (MSCA) Fellowship has had a major impact on the career path of the Fellow, enabling him to secure a permanent academic position as Adjunct Researcher (Senior Lecturer equivalent) in the National Scientific and Technical Research Council in Argentina. The prestige and experience gained from this Fellowship have positioned the Fellow at the forefront of his peers competing for a permanent position. As a newly established independent researcher, the Fellow will seek to secure funds from Argentinian and international public and private organizations, and start attracting talent to lead in his independent research group. The knowledge gained by the Fellow during the Fellowship, as well as the training provided by the Fellow to his colleagues, squarely align with the European strategic priorities for knowledge transfer, and international dimension.

The work carried out in the SPERMAGE project has potential societal implications. While the results of this research were obtained in an insect model, many of the genes underpinning sperm and fertility are conserved across the animal kingdom, making the information broadly transferable, the knowledge gathered through this Fellowship represents a significant leap from the previous state-of-the-art knowledge we had on male fertility deterioration with age. Moreover, these discoveries will provide the necessary proof of concept to pursue further fundamental and applied research in human aging and reproductive health and animal breeding. In particular, the finding of the profound genotype effect would have to be taken into account when designing clinic studies for infertile or sub-fertile men belonging to different populations or ethnic backgrounds. Moreover, the discovery of surprising transgenerational effects of sperm viability on offspring quality is highly meaningful in a European context of increasingly delayed fatherhood. Upon publication, the research outputs will conform with the European Commission’s policy of open-innovation, open-science and open to the world. The high-quality research will strengthen the European seal of excellence, which is part of Pillar 3 – Maximising Impact, of the open-innovation theme. The publications in gold open access will satisfy the open-science theme’s requirements and promote prospective collaborations from around the world.

Drosophila melanogaster sperm stained with fluorochromes that identify live (green) and (dead) cells